Final Report Summary - MICROCOKIT (Microbial Community-based sequencing analysis linked to anthropogenic pressures: MicroCoKit to address the water quality) MicroCokit is a FP7 Marie Curie Industry-Academia Partnerships and Pathways project entitled “Microbial Community-based sequencing analysis linked to anthropogenic pressures: MicroCoKit to address the water quality” and funded under the call FP7-PEOPLE-2012-IAPP. MicroCokit began on 1st of June 2013 and ended up 31 May 2017. It was a close cooperation between academic groups (CNR-IRSA- Italy, EU-JRC, MBA-UK) and leading private enterprises (LGC-UK and NTBC-Spain). The project was coordinated by CNR-IRSA. MicroCokit has been conceived to i) investigate and identify aquatic indicators based on microbial communities linked to anthropogenic pressures and ii) foster the transfer of knowledge among the partners especially through seconded staff from academia to company and vice versa with the final goal to bring to the market faster, sensitive and robust tools to assess the water quality based on quantitative real time PCR (qPCR) and Fluorescence In Situ Hybridization (FISH) techniques. Tiber, the third longest and second largest Italian river, has been chosen as a pilot case study. Four differently anthropogenic influenced areas have been identified for water sampling such as pristine, agriculture, industrial and urban areas to investigate the rate of anthropogenic pressure (e.g. class of chemical pollutants and concentrations) combined with the microbial community characterisation for each area. The community characterisation has been achieved by implementing the most advanced molecular based techniques such as quantitative real time PCR (qPCR), Fluorescence In Situ Hybridization (FISH), Microarray and Metagenomics (full sequencing of water samples) to figure out correlations between the microbial community/microorganisms and group of chemical pollutants present in water. The scientific outcome of this innovative approach was performed in close collaboration with two private enterprises, actively involved in molecular biology diagnostic field, in order to translate it into commercial products and/or kits for water quality assessment. The selection of sampling points, which reflected different sources of contamination, has been the crucial step for achieving the project goal to produce reliable data for identifying the bioindicators. Five sampling campaigns were performed in order to analyse water samples for inorganic contaminants, dissolved organic carbon (DOC), organic contaminants, biological parameters and physical-chemical parameters (e.g. pH, oxygen and temperature). The organic contaminants analysed were Polycyclic Aromatic Hydrocarbon (PAH), pesticides, biocides, pharmaceutical compounds and perfluoroalkyl acids. The microbiological parameters assessed were bacterioplankton abundance, cell viability and phylogenetic characterization of the microbial community using commercial probes for Fluorescence In Situ Hybridization (FISH) analysis.The overall chemical results indicated a correspondence between different groups of contaminants found and the main contamination sources of the selected sampling point areas, such as the absence of chemical pollutants at the river source (pristine); an overall low concentration of pesticides, corrosion inhibitors and PAHs in the agricultural area, together with the presence of some metals due to proximity of a foundry; in the industrial site As and Sr are present owing to the discharge in the river of groundwater naturally containing these metals that is used for the extraction of travertine quarries, and pharmaceuticals are present in this site because it is located nearby a WWTP; finally, corrosion inhibitors, perfluorinated compounds and nutrients are in the urban area corresponding to the river mouth. In the latter sampling point, an antifouling agent was found because this site is also used for boat storage and harbour. PAHs were found in all points where anthropogenic pressures were present, although their concentrations were generally low.The structural microbiological parameters, such as the total microbial abundance and the percentage of the main microbial domains (Bacteria and Archaea) detected in the 4 sampling points, reflected both natural river characteristics (e.g. a general tendency of bacterial abundances to increase from the source to the mouth of the river) and contaminant occurrence. The preliminary results of phylogenetic analysis of the bacterial community revealed differences among the four sampling sites which could reflect their adaptation to the different anthropogenic pressures which will be more deeply investigated by RNA microarray and metagenomics analysis. The attached Figure shows the overall project, the characterisation of microbial communities linked to anthropogenic pressures will be used to identify specific communities/microorganisms as indicators of water quality status (e.g. pristine versus polluted areas). Three different kinds of bioindicators are foreseen, 1) Microbial community based indicator; 2) Pathogen indicator; 3) Ecological bioindicators. The end-products will be a ready to use plate containing defined primers for quantitative Real Time PCR (qPCR) targeting the developed bioindicators and specific probes based on Fluorescence in situ hybridization technique (FISH).Contact details: barracaracciolo@irsa.cnr.it; Teresa.LETTIERI@ec.europa.euThe active address of the project public website is: http://www.microcokit.eu/
